Design, synthesis and in-vitro anti-leukemic evaluation of ferulic … · 2016. 7. 12. · Design, synthesis and in-vitro anti-leukemic evaluation of ferulic acid analogues as Bcr-Abl

Design, synthesis and in-vitro anti-leukemic evaluation of ferulic acid analogues

as Bcr-Abl inhibitors

Narendran Rajendran, Shankar Subramaniam , Mamilla R. Charan Raja, Himesh Makala Venkata

Subbarao, Ulaganathan Venkatasubramanian, Brindha Pemaiah , Santanu Kar Mahapatra* and

Aravind Sivasubramanian*

Electronic supplementary information (ESI)

Materials and methods

Plant collection and extraction

The aerial parts of S. brachiata were collected from the place called killai, near Pichavaram,

Tamil Nadu, and India. A voucher specimen was matched with the authentic specimen

(herbarium no 5555, regional plant resource center, Puri, India) and was deposited in the

herbarium. The aerial parts (~1 kg) were shade dried and powdered. 100 g of the powder was

percolated with Millipore water and after 24 h, the extract were filtered through a Whatman No.

41 filter paper. The extraction was repeated thrice and the combined extracts were concentrated

in vacuo and further lyophilized to obtain a residue (35 g).

Isolation of ferulic acid

10g of water extract of S.brachiata was subjected to column chromatographic separation.

Initially the crude extract was separated by using Sephadex LH 20 (Sigma Aldrich, sweden).

Sephadex was prepared after the swelling time of 24hrs by using chloroform-methanol (30:70)

mixture. There are 18 fractions were collected by increasing polarity of the mobile phase from

30% chloroform: methanol to 100% methanol. The TLC pattern of SB 9 and SB 13 shows

similar in spot nature and they were mixed up accordingly. This fraction reveals two spots in

major (Rf = 0.488, 0.418) and one more with minor intensity. Further purification was done by

using normal silica column (100-200 mesh). Dirty white precipitate was formed along the sides

of the test tube. Further purification of the compound yielded 27mg of ferulic acid. The

structures of the compound have been identified by using NMR Studies.

Procedure for the synthesis of (E)-3-(4-acetoxy-3-methoxyphenyl) acrylic acid (2)

Ferulic acid (19.4g, 0.1 mol) was added to the solution of NaOH (12.7g, 0.26 mol) in 100 mL

water. Then the mixture was stirred and cooled below 10°C. To the cold solution acetic

anhydride (12.7 mL, 0.125 mol) was added in drop wise manner. The reaction mixture was

stirred at 200C for 10min and room temperature for 30min. Then dilute sulfuric acid was added

in dropwise till the white precipitate form. Maintain the pH of the solution 4-5 by using dilute

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2016

sulfuric acid (5N). Filter the white precipitate and washed several times with distilled water. The

product gives white precipitate (15.719 g) with yield of 81%.

General procedure for the synthesis of amide analogues of ferulic acid (2a-2g)

Add 1.2 equivalent Thionyl chloride (86.34 µL, 1.2mmol) to the synthesized (E)-3-(4-acetoxy-3-

methoxyphenyl) acrylic acid (236 mg, 1mmol) and heat the reaction mixture at 800C for 3 hrs.

Remove the free SOCl2 by using vacuum pump and dissolved in dry CH2Cl2.

Amine (1.5 mmol) was dissolved in dry CH2Cl2 followed by adding Trimethylamine (721 µL, 5

mmol) and cool the solution below 100C. To this, acid chloride solution was added dropwise by

using addition funnel. Maintain the temperature of reaction mixture below 100C for 10min and

3hrs in room temperature. The reaction mixture was quenched with water and extracted by using

CH2Cl2 (2 x 20 mL). The organic solution was dried over anhydrous sodium sulfate and solvents

were evaporated using roto vacuum evaporator. The products further purified by subjected to

column chromatography using chloroform/methanol (99:1) as eluent.

Esterification and Thio-esterification of ferulic acid (2h-2k)

Ferulic acid chlorides have been prepared as mentioned in the above procedure (SOCl2 method)

and dissolved this acid chloride in dry CH2Cl2. Thiol (1.5 mmol) was dissolved in dry CH2Cl2

followed by adding Trimethylamine (721 µL, 5 mmol) and cool the solution below 100C. To this,

acid chloride solution was added dropwise by using addition funnel. Maintain the temperature of

reaction mixture below 100C for 10min and 3hrs in room temperature. The reaction mixture was

quenched with water and extracted by using CH2Cl2 (2 x 20 mL). The organic solution was dried

over anhydrous sodium sulfate and solvents were evaporated using roto vacuum evaporator. The

products further purified by subjected to column chromatography using Hexane - ethyl acetate

(80:20) as eluent. The product formed as a white precipitate.

Procedure for the synthesis of methyl- (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (3)

Ferulic acid (194mg, 1mmol) was dissolved in 3mL of methanol followed by adding drops of

Con. H2SO4. Then the mixture was refluxed at 800C for 3hrs. After 3hrs, the mixture was

quenched with water and extracted by using ethyl acetate (2 x 20mL). The organic extract was

separated, washed with bicarbonate, dried over anhydrous sodium sulfate and solvents were

evaporated under reduced pressure. As such of the obtained compound was used for the

synthesis of its respective analogues.

General procedure for O- alkylation of ferulic acid (3a-3l)

Amine (2.5 mmol) was dissolved in dry CH2Cl2 and adds K2CO3 (552 mg, 4 mmol). Stir and

cool the solution below 100C. To this add chloroacetyl chloride (315 µL, 4 mmol) dissolved in

dry CH2Cl2 dropwise by using addition funnel. Maintain the temperature below 100C for 20min

and 3hrs for room temperature. The reaction mixture was quenched with cold water and

extracted with ethyl acetate (2 x 20 mL). The ethyl acetate layer was dried over anhydrous

sodium sulfate and solvents removed under reduced pressure. The acetamide formed as

precipitate and it was subjected to O-alkylation with FA ester.

Take methyl- (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (208 mg, 1mmol) and dissolved

acetonitrile (2mL). Add the acetamide (1.2 mmol) and K2CO3 (165.6 mg, 1.2mmol) to the

reaction mixture. Reflux the reaction mixture at 1200C for 3hrs. After completion of the reaction,

it was quenched with water and extracted with CH2Cl2 (2 x 20 mL). The organic layer then dried

with anhydrous sodium sulfate and solvents were removed under reduced pressure. Product was

purified by column chromatography by using methanol/chloroform (3:97) as eluent.

General procedure for the synthesis of ferulic acid ethers (3m-3w)

Take methyl- (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (208 mg, 1mmol) and dissolved

Dimethyl formamide (2mL). To this add K2CO3 (414 mg, 3mmol) and alkyl bromide (1.5mmol)

and heat the reaction mixture at 800C for 3hrs. After the reaction completes, it was quenched

with water and extracted by using ethyl acetate (3 x 20 mL). The ethyl acetate layer then dried

over with anhydrous sodium sulfate and solvents were removed under reduced pressure. Product

was purified by column chromatography using Hexane – ethyl acetate (95:5) as eluent.

General procedure for the synthesis of 3H-benzo[f]chromen-3-one derivatives from Ferulic

acid (4a-4c)

Ferulic acid (194mg, 1mmol) was taken in 5mL RB flask and mixed with powdered 2-naphthol

(144mg, 1mmol). Add anhydrous AlCl3 (133mg, 1mmol) to the reaction mixture and mixed

thoroughly. Neat reaction was performed at 1200C for 3hrs. After completion of the reaction, the

reaction mixture was quenched with water and extracted with ethyl acetate. Then the organic

layer was dried over with anhydrous sodium sulfate and solvents were removed under reduced

pressure. Product was purified by column chromatography using Hexane – ethyl acetate (80:20)

as eluent.

In silico studies

Ligand Preparation

All the 39 compounds including (E)-4-(3-((4-fluorophenyl)thio)-3-oxoprop-1-en-1-yl)-2-

methoxyphenyl acetate (2i), methyl (E)-3-(3-methoxy-4-(2-oxo-2-

thiomorpholinoethoxy)phenyl)acrylate (3j) along with ferulic acid (1) were drawn by using

ChemSketch and known BCR-ABL tyrosine kinase inhibitors (Imatinib) was retrieved from

Pubchem database. The ligands were subjected to Energy minimization using Avagadro and

subsequently the charges were added to them through Autodock 4.0.

Protein preparation

The crystal structure of the target protein BCR-ABL tyrosine kinase1 (PDB ID: - 3OXZ) was

retrieved from PDB database (http://www.rcsb.org/pdb) having resolution 2.2 Å. All the

heteroatoms and water molecules were removed by Discovery Analyzer software. Polar

hydrogens and Gasteiger charges were assigned after merging of non-polar hydrogen atoms in

ADT.

Molecular docking

To validate drug-target association, the molecular docking stimulation was performed on ligands

with BCR-ABL tyrosine kinase by Autodock software 4.0 by employing Lamarckian genetic

algorithm2. The receptor was kept rigid, while ligands were set flexible to rotate and explore

most probable binding poses. The torsional bonds of ligands were set free by Ligand module in

AutoDock Tool (ADT). As per earlier reports, the Grid was set by covering active site binding

pocket of the protein (chain A) with 92×92×92 points in x, y, z direction and followed by

Autogrid run. The docking protocol was then implemented on the ligands in the dataset. The

study was carried for 20 GA runs, which was found to be optimum to reproduce the pose in its

crystal. The other GA parameters like the population size and the genetic operators were kept at

their default values. The results were interpreted by observing the least binding energy

(kcal/mol) and hydrogen bonds; stacking interactions involving between the active site residues

of the target and ligand molecules were analyzed using PyMol molecular visualization tool and

LigPlot. The RMSD was calculated to ensure that the selected ligands have conformational

similarity.

Anti-leukemic activity of FA derivatives

In vitro cell viability assay

All cell lines were procured from the cell repository of the National Centre for Cell Science,

Pune, India. The dose and duration dependent cytotoxicity of Ferulic acid and its derivatives on

PBML, K562, U937 and HepG2 cell lines were quantitatively estimated by a non-radioactive,

colorimetric assay system using tetrazolium salt, 3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenil-

tetrazolium bromide (MTT). The percentage of proliferation was calculated by using the

following equation:

% Proliferation = [OD sample – OD control] X 100/OD control

The concentration required for a 50% inhibition of viability (IC50) was determined graphically.

Multiple linear regressions were used to compare data using GraphPad prism 6 software. Among

all the derivatives of FA, 2i and 3j were found as most active compounds and subjected into

further biochemical studies.

Intracellular measurement of reactive oxygen species (ROS)

The production of intracellular ROS was measured using 2,7- dichlorofluorescein diacetate

(DCFH2-DA) [43]. To the stock solution (in methanol) of 10 mM DCFH2-DAwas diluted in

http://www.rcsb.org/pdb

culture medium without serum or other additives to yield a 100 μM working solution. At the end

of exposure with Ferulic acid, 2i and 3j and the cells were washed twice with PBS. Then, cells

were incubated in 1.5 mL working solution of DCFH2-DA at 37oC for 30 min. Cells were lysed

in alkaline solution and centrifuged at 3000 rpm. One milliliter of supernatant was transferred to

a cuvette, and fluorescence was measured at 520 nm with a fluorescence spectrophotometer

(Jasco-FP8200) using 485-nm excitation. As a positive control, those cells were incubated with

H2O2 (100 mM) for 30 min prior to the analysis. The values were expressed as percent

fluorescence intensity relative to control wells. Fluorescence micrographs were also taken by

fluorescence microscopy (Carl Zeiss Axio Scope A1). To determine the crucial cues of ROS

generation in shikonin derivatives-induced cell death, K562 cells (2×104) were seeded in a 96-

well plate with 0.2 mL per well. A stock solution of N-acetyl-L-cysteine (NAC) was made with

sterile water and added to cells at 10 mM for 1 h [42]. NAC pretreated or untreated K562 cells

were cultured with 100 nM FA derivatives for 24 h and cell viability was determined by the

MTT method.

Determination of reduced glutathione (GSH)

Reduced glutathione estimation in the cell lysate was performed by the method of Moron et al.

The required amount of the cell lysate was mixed with 25% of trichloroacetic acid and

centrifuged at 2,000 xg for 15 min to settle the precipitated proteins. The supernatant was

aspirated and diluted to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6

mM DTNB was added. After 10 minutes the optical density of the yellow-colored complex

formed by the reaction of GSH and DTNB (Ellman’s reagent) was measured at 405 nm. A

standard curve was obtained with standard reduced glutathione. The levels of GSH were

expressed as μg of GSH/mg protein.

Determination of oxidized glutathione (GSSG)

The oxidized glutathione level was measured after derivatization of GSH with 2-vinylpyidine

according to the method of Griffith et al. In brief, with 0.5 mL cell lysate, 2 μl of 2-vinylpyidine

was added and incubates for 1 hr at 37°C. Then the mixture was deprotenized with 4%

sulfosalicylic acid and centrifuged at 1,000 xg for 10 min to settle the precipitated proteins. The

supernatant was aspirated and GSSG level was estimated with the reaction of DTNB at 412 nm

in spectrophotometer and calculated with standard GSSG curve.

Redox ratio (GSH/GSSG)

Redox ratio was determined for FA, 2i and 3j by taking the ratio of reduced glutathione/oxidized

glutathione.

Determination of lipid peroxidation (MDA)

Lipid peroxidation was estimated by the method of Ohkawa et al. in cell lysate. The reaction

mixture contains Tris-HCl buffer (50 mM, pH 7.4), tert-butyl hydroperoxide (BHP) (500 μM in

ethanol) and 1 mM FeSO4. After incubating the samples at 37°C for 90 min, the reaction was

stopped by adding 0.2 mL of 8% sodium dodecyl sulfate (SDS) followed by 1.5 mL of 20%

acetic acid (pH 3.5). The amount of malondialdehyde (MDA) formed during incubation was

estimated by adding 1.5 mL of 0.8% thiobarbituric acid (TBA) and further heating the mixture at

95°C for 45 min. After cooling, samples were centrifuged, and the thiobarbituric acid reactive

substances (TBARS) were measured in supernatants at 532 nm by using 1.53 x 105 M-1 cm-1 as

extinction coefficient. The levels of lipid peroxidation were expressed in terms of n mol/mg

protein.

Quantitative estimation of DNA fragmentation by diphenylamine (DPA) assay

After the treatment schedule, cells were lysed with hypotonic lysis buffer followed by

centrifugation. Then Perchloric acid (0.5 M) was added to the pellets containing uncut DNA

(resuspended with 200 mL of hypotonic lysis buffer), and to the other half of the supernatants

containing DNA fragments. Then 2 volumes of a solution containing 0.088 M DPA, 98% (v/v)

glacial acetic acid, 1.5% (v/v) sulfuric acid, and 0.5% (v/v) of 1.6% acetaldehyde solution were

added. The samples were stored at 4 ºC for 48 h. The colorimetric reaction was quantitated

spectrophotometrically at 575 nm. The percentage of fragmentation was calculated as the ratio of

DNA in the supernatants to the total DNA confirm the data obtained from the spectrophotometric

method, we have analyzed the DNA fragmentation by DNA laddering in 1.2% agarose gel

electrophoresis.

Apoptotic morphological changes by AO/Et-Br staining

Two DNA-binding dyes AO and Et-Br were used for the morphological apoptotic cells. After

treatment with FA derivatives for 24 h, K562 cells were collected, washed with cold PBS and

then stained with a mixture of AO (100 μg/mL) and Et-Br (100 μg/mL) at room temperature for

5 min in dark. After proper washing with PBS, the stained cells were observed by a fluorescence

microscope (Carl Zeiss Axio Scope A1). Untreated and FA derivatives treated K562 cells were

collected and were fixed with 2.5% glutaraldehyde for 15 min, followed by permeabilized with

0.1% Triton X-100 and stained with 1 μg/mL DAPI for 5 min at 37oC. The cells were then

washed with PBS and examined by fluorescence microscopy (Carl Zeiss Axio Scope A1).

Bcr-Abl kinase inhibitory assay - ADP-Glo™ Kinase assay kit

The Bcr–Abl inhibitory activity assay was performed using ADP-Glo™ Kinase assay kit

(Promega, USA) and Abl Kinase Enzyme System (Promega, USA) according to the

manufacturer’s instructions. General procedures are as the following: Kinases (4ng/μL) were

incubated with substrates (0.2μg/μL), compounds (3×10-5

- 3×10-10

M) and ATP (25μM) in a

final buffer of tris 40mM, MgCl2 10mM, BSA 0.1mg/mL, DTT 1mM in 384-well plate with the

total volume of 5μL. The assay plate was incubated at 30ºC for 1h. After the plate cooled for

5min at room temperature, 5μl of ADP-Glo reagent was added into each well to stop the reaction

and consume the remaining ADP within 40min. At the end, 10μL of kinase detection reagent was

added into the well and incubated for 30min to produce a luminescence signal and the

luminescence was measured with the help of a plate-reading luminometer. The signal was

correlated with the amount of ATP present in the reaction and was inversely correlated with the

kinase activity.

Assessment of apoptotic cell population by annexin V-FITC/PI assay

Confluent culture of K562 cells (2 × 106) were seeded in 6 well cell culture plates and treated

with FA, 2i, 3j for 24 h. Then the K562 cells were collected, washed with PBS, and re-suspended

in PBS. Apoptotic cell death was identified by double staining with annexin V-FITC and PI,

using the annexin V-FITC apoptosis detection kit (BD Bioscience) according to the

manufacturer's instructions. Data acquisition and analysis were performed in a Becton-Dickinson

FACS verse flow cytometer using CellQuest software.

BCR-Abl mRNA expression study:

Total RNA extracted from untreated and treated K562 cells after 6 hrs (TRIZOL; Invitrogen). 1

μg of total RNA were reverse transcribed using Revert Aid M-MuLV Reverse Transcriptase

(Fermentas) to synthesis the cDNA. cDNA was amplified with green TaqDNA polymerase

(Fermentas) in 25 μL reaction mixture using specific primers of bcr-abl and gapdh using Veriti

Thermal Cycler (Applied Biosystems). Sequences of the PCR primers were as follows: bcr-abl

product representing the BCR-ABL junction and kinase, forward: 5'-GAA GCT TCT CCC TGG

CAT CCC GT-3' and reverse 5'-GCC AGG CTC TCG GGT GCA GTC C-3'; gapdh, forward 5'-

GAG CCA AAC GGG TCA TCA TC-3' and reverse, 5'-CCT GCT TCA CCA CCT TCT TG-3'.

The reaction conditions consisted of an initial activation step (5 min at 95°C) and a cycling step

(denaturation for 30 s at 94°C, annealing for 30 s at 58°C and extension for 1 min at 72°C for 35

cycles). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification was also done to

ensure equal cDNA input. PCR-amplified product was subsequently run on 1.2% agarose gel,

stained with ethidium bromide and visualized under ultraviolet light and analyzed in BioRad gel

documentation system. In real time PCR, (Eppendorf RealPlex master cycler, using 2X SYBR

premix Ex Taq II (TAKARA Bio), the mRNA expression levels of the target genes were

normalized against those of GAPDH levels and expressed as relative fold change compared with

untreated controls and quantified by the 2–ddCT

method.

Fig. S1 High Performance Thin Layer Chromatography (HPTLC) profile of Standard Ferulic

acid (Lane 1) compared with aqueous extract of Salicornia brachiata (Lane 2 and 3).

Fig. S2 Compounds synthesized by using –OH Protected ester of ferulic acid

Fig. S3 Compounds synthesized by using Methyl ferulate

Fig. S4 Crystal structure of the product (a) Structure of 4a, (b) Crystal structure of 4a [CCDC 1449366], (c) Structure of 3g and (d) Crystal structure of 3g [CCDC 1454627].

Fig. S5 LC Chromatogram and mass spectrum of compound 2h

Fig. S6 LC Chromatogram and mass spectrum of compound 2i

Fig. S7 LC Chromatogram and mass spectrum of compound 2j

Fig. S8 LC Chromatogram and mass spectrum of compound 3j

Fig S9. DNA Fragmentation studies of FA, 2i, 3j and imatinib

Characterization of the compounds

(E)-3-(4-hydroxy-3-methoxyphenyl) acrylic acid (1)

Mp: 169-171oC; MF: C10H10O4; Appearance: white solid;

1H NMR (300 MHz, d6-DMSO): 12.13

(s, 1H), 9.56 (s, 1H), 7.48 (d, J=15.9Hz, 1H), 7.28 (d, J=1.8Hz, 1H), 7.08 (dd, J=1.8Hz, 6.3Hz,

1H), 6.78 (d, J=8.1Hz, 1H), 6.36 (d, J=15.9Hz, 1H), 3.81 (s, 3H); 13

C NMR (300 MHz, d6-

DMSO): 167.98, 149.02, 147.86, 144.49, 125.73, 122.79, 115.57, 115.47, 111.06, 55.61; LC MS

(m/z): [M + H+ ]- 194.19

Methyl (E)-3-(4-acetoxy-3-methoxyphenyl) acrylate (2)

Yield : 89%; Mp: 197-199oC (Reference 197

oC); MF: C13H14O5; Appearance: white solid;

1H

NMR (300 MHz, d6-DMSO): 12.46 (s, 1H), 7.57 (d, J=15.9Hz, 1H), 7.48 (d, J=1.8Hz, 1H), 7.26

(dd, J=1.8Hz, 6.6Hz, 1H), 7.11 (d, J=8.1Hz, 1H), 6.59 (d, J=15.9Hz, 1H), 3.82 (s, 3H), 2.264 (s,

3H); ; LC MS (m/z): [M + H+ ]- 250.25

(E)-3-(4-hydroxy-3-methoxyphenyl)-N, N-dimethyl acrylamide (2a)

Yield : 45%; Mp: 116-118oC; MF: C12H15NO3; Appearance: white solid;

1H NMR (300 MHz,

CDCl3): 7.60 (d, J=15.3Hz, 1H), 7.10 (dd, J=1.8Hz, 6.3Hz, 1H), 6.99 (d, J=1.8Hz, 1H), 6.91 (d,

J=8.1Hz, 1H), 6.73 (d, J=15.6Hz, 1H), 5.91 (s, 1H), 3.93(s, 3H), 3.17(s, 3H), 3.07(s, 3H); 13

C

NMR (300 MHz, d6-DMSO): 167.04, 147.35, 146.74, 142.60, 127.87, 121.81, 114.78, 110.05,

55.98, 37.45, 35.99; ; LC MS (m/z): [M + H+ ]- 221.26

(E)-4-(3-(diethylamino)-3-oxoprop-1-en-1-yl)-2-methoxyphenyl acetate (2b)


1H NMR (300 MHz,

CDCl3): 7.66 (d, J=15.3Hz, 1H), 7.14 (d, J=8.1Hz, 1H), 7.05 (d, J=3.9Hz, 1H), 7.02 (s, 1H), 6.75

(d, J=15.3Hz, 1H), 3.8 (s, 3H), 3.53-3.44 (m, 4H), 2.32 (s, 3H), 1.28-1.17 (m, 6H); 13

C NMR

(300 MHz, d6-DMSO): 168.88, 165.49, 151.21, 141.61, 140.64, 134.56, 123.06, 120.05,118.15,

111.79, 55.91, 42.28,41.06, 20.63, 15.05, 13.18; ; LC MS (m/z): [M + H+ ]- 291.15

(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(pyrrolidin-1-yl) prop-2-en-1-one (2c)


1H NMR (300 MHz,

CDCl3): 7.66 (d, J=15.3Hz, 1H), 7.11 (dd, J=1.8Hz, 6.6Hz,1H), 7.0 (d, J=1.8Hz, 1H), 6.91 (d,

J=8.1Hz, 1H), 6.57 (d, J=15.6Hz, 1H), 5.88 (s, 1H), 3.93 (s, 3H), 3.65-3.57 (m, 4H), 2.05-1.85

(m, 4H); 13

C NMR (300 MHz, CDCl3)165.06, 147.41, 146.74, 141.91, 127.80, 121.82, 116.19,

114.78, 110.11, 55.94, 46.58, 46.04, 26.11, 24.34; LC MS (m/z): [M + H+ ]- 247.12

(E)-2-methoxy-4-(3-oxo-3-(piperidin-1-yl) prop-1-en-1-yl) phenyl acetate (2d)


1H NMR (300 MHz,

CDCl3): 7.52 (d, J=15.6Hz, 1H), 7.05 (d, J=8.4Hz,1H), 6.97 (t, J=8.1Hz,8.4Hz, 2H), 6.76 (d,

J=15.3Hz, 1H), 3.79 (s, 3H), 3.59 (s, 2H), 3.51 (s, 2H), 2.25 (s, 3H), 2.1 (s, 1H), 1.63-1.54 (m,

5H); 13

C NMR (300 MHz, CDCl3) 168.88, 165.16, 151.26, 141.49, 140.67, 134.56, 123.08,

120.23, 118.08, 111.53, 55.96, 47.06, 43.36, 26.78, 25.6, 24.63, 20.66;; LC MS (m/z): [M + H+

]- 303.15

(E)-3-(4-hydroxy-3-methoxyphenyl)-1-morpholinoprop-2-en-1-one (2e)


1H NMR (300 MHz,

CDCl3): 7.56 (d, J=15.3Hz, 1H), 7.04-7,01 (m,1H), 6.91 (d, J=1.8Hz,1H), 6.84 (d, J=8.4Hz,

1H), 6.61 (d, J=15.3Hz,1H), 5.90 (s, 1H), 3.86 (d, J=3.6Hz, 3H), 3.65 (s, 8H); 13

C NMR (300

MHz, CDCl3) 165.93, 147.58, 146.81, 143.52, 127.60, 121.99, 114.85, 113.79, 109.97, 66.88,

55.99, 46.26, 42.46, 29.69; ; LC MS (m/z): [M + H+ ]- 263.12

(E)-4-(3-(benzylamino)-3-oxoprop-1-en-1-yl)-2-methoxyphenyl acetate (2f)


1H NMR (300 MHz,

CDCl3): 7.55 (d, J=15.6Hz, 1H), 7.30-7.18 (m, 6H), 7.03-6.33(m, 3H), 6.27 (d, J=15.6Hz, 1H),

5.88 (s, 1H), 4.50 (d, J=5.7Hz, 1H), 3.7 (s, 3H), 2.2 (s, 3H); 13

C NMR (300 MHz, CDCl3)

168.95, 165.59, 151.27, 140.89, 140.64, 138.12, 133.84, 128.76, 127.94, 127.85, 127.61, 123.15,

120.76, 120.56, 111.40, 55.87, 43.89, 20.66; ; LC MS (m/z): [M + H+ ]- 325.13

(E)-2-methoxy-4-(3-oxo-3-(phenylthio) prop-1-en-1-yl) phenyl acetate (2h)

Yield : 65%; Mp: 119-121oC; MF: C18H16O4S; Appearance: white solid;

1H NMR (300 MHz,

CDCl3): 7.63 (d, J=15.9Hz, 1H), 7.51-7.43 (m, 5H), 7.15 (td, J=1.8Hz, 2H), 7.07 (d, J=8.1Hz,

1H), 6.73 (d, J=15.6Hz, 1H), 3.88 (s, 3H), 2.33 (s, 3H); 13

C NMR (300 MHz, CDCl3) 187.76,

168.70, 151.50, 141.83, 140.77, 134.62, 132.95, 129.22, 127.52, 124.30, 123.38, 121.69, 111.59,

55.97, 20.64; ; LC MS (m/z): [M + H+ ]- 328.08.

(E)-4-(3-((4-fluorophenyl) thio)-3-oxoprop-1-en-1-yl)-2-methoxyphenyl acetate (2i)

Yield : 70%; Mp: 108-110oC; MF: C18H15FO4S; Appearance: white solid;

1H NMR (300 MHz,

CDCl3): 7.63 (d, J=15.6Hz, 1H), 7.48-7.43 (m, 2H), 7.19-7.11 (m, 4H), 7.08 (d, J=8.4Hz, 1H),

6.72 (d, J=15.9Hz, 1H), 3.89 (s, 3H), 2.33 (s, 3H); 13

C NMR (300 MHz, CDCl3) 187.76, 168.73,

151.53, 141.07, 136.78, 136.66, 132.86, 124.04, 123.43, 122.80, 122.75, 121.75, 116.68, 116.38,

111.59, 55.99, 20.66; ; LC MS (m/z): [M + H+ ]- 346.07

(E)-2-methoxy-4-(3-oxo-3-(p-tolylthio) prop-1-en-1-yl) phenyl acetate (2j)

Yield : 40%; Mp: 153-155oC; MF: C19H18O4S; Appearance: white solid;

1H NMR (300 MHz,

CDCl3): 7.64 (d, J=15.9Hz, 1H), 7.61 (d, J=1.8Hz, 1H), 7.39-7.36 (m, 3H), 7.30 (d, J=8.4Hz

,2H), 7.21 (s, 1H), 7.16 (t, J=9.3,1H), 3.84 (s, 3H), 2.36 (s, 3H), 2.27 (s, 3H); 13

C NMR (300

MHz, CDCl3) 187.32, 168.32, 151.20, 141.39, 140.73, 139.40, 134.46, 132.67, 129.96, 124.48,

123.70, 123.30, 122.35, 112.27, 56.04, 20.81, 20.37; ; LC MS (m/z): [M + H+ ]- 342.09

Phenyl- (E)-3-(4-acetoxy-3-methoxyphenyl) acrylate (2k)

Yield : 57%; Mp: 162-163oC; MF: C18H16O5; Appearance: white solid;

1H NMR (300 MHz,

CDCl3): 7.83 (d, J=16.2Hz, 1H), 7.44-7.38 (m, 2H), 7.287-7.16 (m, 5H), 7.09 (d, J=8.1, 1H),

6.58 (d, J=16.2Hz, 1H), 3.84 (s, 3H), 2.54 (s, 3H); 13

C NMR (300 MHz, CDCl3) 168.72, 165.21,

151.45, 150.72, 145.77, 141.73, 133.08, 129.43, 125.80, 123.23, 121.57, 121.45, 117.49, 111.38,

55.90, 20.62; ; LC MS (m/z): [M + H+ ]- 312.10

Methyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (3)

Yield: 88%; MF: C11H12O4; Appearance: Brown viscus liquid; 1H NMR (300 MHz, CDCl3):

7.62 (d, J=15.9Hz, 1H), 7.04 (td, J=1.8Hz, 2H), 6.91 (d, J=8.1Hz, 1H), 6.29 (d, J=15.9Hz, 1H),

3.91 (s, 3H), 3.79 (s, 3H); ; LC MS (m/z): [M + H+ ]- 208.07

Methyl (E)-3-(3-methoxy-4-(2-oxo-2-(phenylamino)ethoxy)phenyl)acrylate (3a)

Yield: 51%; Mp: 148-150oC; MF: C19H19NO5; Appearance: White solid;

1H NMR (300 MHz,

CDCl3): 8.76 (s, 1H), 7.66-7.57 (m, 3H), 7.38-7.33 (m, 2H), 7.18-7.10 (m, 3H), 6.96 (d,

J=8.1Hz, 1H), 6.35 (d, J=15.9Hz, 1H), 4.67 (s, 2H), 3.97 (s, 3H), 3.81 (s, 3H); 13

C NMR (300

MHz, CDCl3) 167.30. 166.18, 149.84, 148.79, 144.05, 137.00, 129.76, 129.03, 124.69, 122.22,

119.86, 116.88, 115.60, 110.57, 69.68, 55.91, 51.66; ; LC MS (m/z): [M + H+ ]- 341.13

Methyl (E)-3-(4-(2-((4-chlorophenyl) amino)-2-oxoethoxy)-3-methoxyphenyl)acrylate (3b)

Yield: 48%; Mp: 188-190oC; MF: C19H18ClNO5; Appearance: White solid;

1H NMR (300 MHz,

CDCl3): 8.80 (s, 1H), 7.63 (d, J=15.9Hz, 1H), 7.58-7.53 (m, 2H), 7.34-7.28 (m, 2H), 7.15-7.10

(m, 2H), 6.96 (d, J=8.1Hz, 1H), 6.36 (d, J=15.9Hz, 1H), 4.66 (s, 2H), 3.97 (s, 3H), 3.81 (s, 3H); 13

C NMR (300 MHz, CDCl3) 167.33. 166.28, 149.89, 148.72, 144.02, 135.64, 130.00, 129.74,

124.12, 122.29, 121.08, 117.08, 115.84, 110.68, 69.77, 56.01, 51.74; ; LC MS (m/z): [M + H+ ]-

375.09

Methyl (E)-3-(3-methoxy-4-(2-((4-nitrophenyl)amino)-2-oxoethoxy)phenyl)acrylate (3d)

Yield: 53%; Mp: 178-180oC; MF: C19H18N2O7; Appearance: pale yellow solid;

1H NMR (300

MHz, d6-DMSO): 10.66 (s, 1H), 8.65 (t, J=2.1Hz, 1H), 7.98-7.93 (m, 2H), 7.66-7.57 (m, 2H),

7.42 (d, J=1.8Hz, 1H), 7.26 (dd, J=1.8Hz, 6.6Hz, 1H), 6.96 (d, J=8.4Hz, 1H), 6.61 (d, J=15.9Hz,

1H), 4.81 (s, 2H), 3.81 (s, 3H), 3.71 (s, 3H); 13

C NMR (300 MHz, d6-DMSO) 167.16. 166.87,

149.37, 149.23, 147.92, 144.45, 139.49, 130.24, 127.96, 125.43, 122.48, 118.17, 115.88, 113.62,

113.57, 111.15, 67.82, 55.94, 51.30; ; LC MS (m/z): [M + H+ ]- 386.11

Methyl (E)-3-(3-methoxy-4-(2-((3-methoxyphenyl) amino)-2-oxoethoxy) phenyl)acrylate

(3e)


1H NMR (300 MHz,

CDCl3): 8.75 (s, 1H), 7.61 (d, J=15.9Hz, 1H), 7.34 (t, J=2.1Hz, 2.4Hz, 1H), 7.27-7.22 (m, 1H),

7.14 (d, J=1.8Hz, 2H), 7.11-7.04 (m, 1H), 6.93 (d, J=8.4Hz, 1H), 6.72-6.68 (m, 1H), 6.36 (d,

J=15.9Hz, 1H), 4.66 (s, 2H), 3.97 (s, 3H), 3.81 (d, J=3.6, 6H); 13


167.37. 166.26, 160.21, 149.93, 148.86, 144.11, 138.28, 129.89, 129.78, 122.29, 116.99, 115.78,

112.07, 110.43, 105.79, 69.83, 56.01, 55.34, 51.74, 29.70; ; LC MS (m/z): [M + H+ ]- 371.14

Methyl (E)-3-(3-methoxy-4-(2-oxo-2-(o-tolylamino)ethoxy) phenyl)acrylate (3f)

Yield: 42%; Mp: 157-160oC; MF: C20H21NO5; Appearance: Grey solid;

1H NMR (300 MHz,

CDCl3): 10.03 (s, 1H), 7.62 (d, J=15.9Hz, 1H), 7.45-7.37 (m, 3H), 7.26-7.17 (m, 2H), 6.94 (d,

J=8.4Hz, 1H), 6.89 (d, J=7.8Hz, 1H) ,6.52 (d, J=15.9Hz, 1H), 4.74 (s, 2H), 3.85 (s, 3H), 3.71 (s,

3H), 2.27 (s, 3H); 13

C NMR (300 MHz, CDCl3) 166.88. 166.13, 149.51, 144.48, 138.25, 137.98,

128.60, 127.82, 124.33, 122.50, 119.88, 116.55, 115.80, 113.48, 110.11, 67.93, 55.74, 51.28,

21.12; ; LC MS (m/z): [M + H+ ]- 355.19

Methyl (E)-3-(4-(2-(benzylamino)-2-oxoethoxy)-3-methoxyphenyl) acrylate (3g)


1H NMR (300 MHz,

CDCl3): 8.50 (t, J=6Hz, 5.7Hz, 1H), 7.60 (d, J=15.9Hz, 1H), 7.40 (, 1H), 7.33-7.22 (m, 6H),

6.93 (d, J=8.4Hz, 1H), 6.60 (d, J=15.9Hz, 1H), 4.613 (s, 2H), 4.33 (d, J=6Hz, 2H), 3.817 (s,

3H), 3.717 (s, 3H); ; LC MS (m/z): [M + H+ ]- 355.14

Methyl (E)-3-(4-(2-(diphenylamino)-2-oxoethoxy)-3-methoxyphenyl) acrylate (3h)


1H NMR (300 MHz,

CDCl3): 7.61 (d, J=15.9Hz, 1H), 7.38-7.26 (m, 10H), 7.06(t, J=1.8Hz, 8.4Hz, 2H), 6.78 (d,

J=7.8Hz, 1H), 6.31 (d, J=15.9Hz, 1H), 4.66 (s, 2H), 3.86 (s, 3H), 3.79 (s, 3H); 3C NMR (300

MHz, CDCl3) 167.62. 167.03, 149.64, 149.58, 144.66, 129.64, 128.42, 122.14, 115.90, 113.44,

110.56, 67.54, 55.93, 51.64; LC MS (m/z): [M + H+ ]- 417.16

Methyl (E)-3-(3-methoxy-4-(2-morpholino-2-oxoethoxy) phenyl) acrylate (3i)


1H NMR (300 MHz,

CDCl3): 7.62 (d, J=16.2Hz, 1H), 7.09 (t, J=1.8Hz, 8.1Hz, 2H), 6.93 (d, J=8.1Hz, 1H), 6.32 (d,

J=8.1Hz, 1H), 4.79 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H); 3.64 (d, J=3.9Hz, 8H), 13

C NMR (300

MHz, CDCl3) 167.47. 166.10, 149.51, 148.98, 144.39, 128.70, 122.22, 116.21, 113.53, 110.32,

68.42, 66.76, 55.83, 51.63, 45.91, 42.51; ; LC MS (m/z): [M + H+ ]- 335.14

Methyl (E)-3-(3-methoxy-4-(2-oxo-2-thiomorpholinoethoxy) phenyl) acrylate (3j)

Yield: 52%; Mp: 110-112oC; MF: C17H21SNO5; Appearance: White solid;

1H NMR (300 MHz,


J=15.9Hz, 1H), 4.77 (s, 2H), 3.92-3.825 (m, 10H), 2.67-2.58 (m, 4H), 13

C NMR (300 MHz,

CDCl3) 167.48. 166.10, 149.44, 149.02, 144.40, 128.68, 122.23, 116.21, 113.34, 110.29, 68.59,

55.82, 51.63, 48.34, 44.78, 27.94, 27.34; ; LC MS (m/z): [M + H+ ]- 351.13.

Methyl (E)-3-(3-methoxy-4-(2-oxo-2-(piperidin-1-yl) ethoxy) phenyl) acrylate (3k)


1H NMR (300 MHz,


J=15.9Hz, 1H), 4.78 (s, 2H), 3.92 (s, 3H), 3.80 (s, 3H), 3.54-3.49 (m, 4H), 1.66-1.60 (m, 5H),

1.25 (s, 1H), 13

C NMR (300 MHz, CDCl3) 167.56. 165.65, 149.53, 144.60, 128.31, 122.33,

115.91, 113.37, 110.34, 68.29, 55.90, 51.60, 46.33, 43.26, 29.66, 26.41, 25.52, 24.41; ; LC MS

(m/z): [M + H+ ]- 333.16

Methyl (E)-3-(3-methoxy-4-(2-oxo-2-(pyrrolidin-1-yl) ethoxy)phenyl)acrylate (3l)


1H NMR (300 MHz,

CDCl3): 7.63 (d, J=15.9Hz, 1H), 7.08 (t, J=2.1Hz, 7.2Hz, 2H), 6.91 (t, J=1.2Hz, 7.5Hz, 1H), 6.30

(d, J=15.9Hz, 1H), 4.72 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 3.56-3.49 (m, 4H), 2.01-1.80 (m, 4H);

LC MS (m/z): [M + H+ ]- 319.20

Methyl (E)-3-(4-ethoxy-3-methoxyphenyl) acrylate (3m)

Yield: 61%; Mp: 94-96oC; MF: C13H16O4; Appearance: White solid;

1H NMR (300 MHz,

CDCl3): 7.64 (d, J=15.9Hz, 1H), 7.10-7.04 (m, 2H), 6.85 (d, J=8.1Hz, 1H), 6.32 (d, J=15.9Hz,

1H), 4.13 (q, J=7.4Hz,6.9Hz, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 1.48 (t, J=6.9Hz, 7.2Hz, 3H); 13

C

NMR (300 MHz, CDCl3) 167.70, 150.52, 149.39, 144.85, 127.18, 122.57, 115.33, 112.13,

109.92, 64.35, 55.92, 51.59, 14.68; ; LC MS (m/z): [M + H+ ]- 236.10

Methyl (E)-3-(3-methoxy-4-propoxyphenyl) acrylate (3n)


1H NMR (300 MHz,


1H), 4.01 (t, J=5.4Hz, 6.9Hz, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 1.94-1.82 (m, 2H),1.045 (t,

J=7.5Hz, 7.2Hz, 3H); 13

C NMR (300 MHz, CDCl3) 167.67, 150.76, 149.48, 144.84, 127.12,

122.54, 115.26, 112.32, 110.11, 70.41, 55.96, 51.55, 22.33, 10.35; ; LC MS (m/z): [M + H+ ]-

250.12

Methyl (E)-3-(4-butoxy-3-methoxyphenyl) acrylate (3o)


1H NMR (300 MHz,


1H), 4.05 (t, J=6.9Hz, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 1.89-1.79 (m, 2H), 1.56-1.43 (m, 2H),

1.045 (t, J=7.5Hz, 7.2Hz, 3H); 13

C NMR (300 MHz, CDCl3) 167.66, 150.79, 149.47, 144.83,

127.09, 122.53, 115.23, 112.28, 110.09, 68.64, 55.95, 51.53, 31.04,19.13,13.91; ; LC MS (m/z):

[M + H+ ]- 264.14

Methyl (E)-3-(3-methoxy-4-(pentyloxy) phenyl) acrylate (3p)


1H NMR (300 MHz,


1H), 4.04 (t, J=6.9Hz, 2H), 3.89 (s, 3H), 3.79 (s, 3H), 1.90-1.816 (m, 2H), 1.49-1.32 (m, 4H),

0.98 (t, J=7.2Hz, 7.5Hz, 3H); 13

C NMR (300 MHz, CDCl3) 167.62, 150.75, 149.43, 144.80,

127.06, 122.50, 115.20, 112.24, 110.05, 68.91, 55.91, 51.49, 28.17, 27.99, 22.37, 13.91; ; LC

MS (m/z): [M + H+ ]- 278.15

Methyl (E)-3-(4-(hexyloxy)-3-methoxyphenyl) acrylate (3q)


1H NMR (300 MHz,


1H), 4.04 (t, J=6.9Hz, 2H), 3.89 (s, 3H), 3.79 (s, 3H), 1.90-1.80 (m, 2H), 1.50-1.41 (m, 2H),

1.36-1.28 (m, 4H), 0.90 (t, J=7.5Hz, 7.2Hz, 3H); 13

C NMR (300 MHz, CDCl3) 167.68, 150.78,

149.46, 144.85, 127.09, 122.55, 115.23, 112.28, 110.07, 68.98, 55.95, 51.55, 31.52, 28.96, 25.56,

22.53, 13.97; ; LC MS (m/z): [M + H+ ]- 292.17

Methyl (E)-3-(4-(heptyloxy)-3-methoxyphenyl) acrylate (3r)


1H NMR (300 MHz,

CDCl3): 7.63 (d, J=15.9Hz, 1H), 7.07 (dd, J=1.8Hz, 2H), 6.85 (d, J=8.1Hz, 1H), 6.31 (d,

J=15.9Hz, 1H), 4.04 (t, J=6.9Hz, 6.6Hz, 2H), 3.89 (s, 3H), 3.79 (s, 3H), 1.90-1.80 (m, 2H), 1.49-

1.25 (m, 8H), 0.88 (q, J=2.1Hz, 4.5Hz, 3H); 13

C NMR (300 MHz, CDCl3) 167.72, 150.82,

149.50, 144.89, 127.13, 122.58, 115.26, 112.58, 110.10, 69.02, 55.98, 51.59, 31.74, 29.03, 25.89,

22.59, 14.09; ; LC MS (m/z): [M + H+ ]- 306.18

Methyl (E)-3-(3-methoxy-4-(octyloxy)phenyl)acrylate (3s)


1H NMR (300 MHz,

CDCl3): 7.63 (d, J=15.9Hz, 1H), 7.09 (d, J=1.8Hz, 1H), 7.07-7.043 (m, 1H), 6.85 (d, J=8.4Hz,

1H), 6.30 (d, J=15.9Hz, 1H), 4.04 (t, J=6.9Hz, 2H), 3.89 (s, 3H), 3.79 (s, 3H), 1.90-1.80 (m, 2H),

1.47-1.28 (m, 10H), 0.90-0.86 (m, 3H); 13

C NMR (300 MHz, CDCl3) 167.71, 150.82, 149.50,

144.88, 127.12, 122.58, 115.26, 112.31, 110.10, 69.01, 55.97, 51.58, 31.79, 29.33, 29.20, 29.03,

25.92, 22.64, 14.09; ; LC MS (m/z): [M + H+ ]- 320.30

Methyl (E)-3-(4-(allyloxy)-3-methoxyphenyl) acrylate (3t)


1H NMR (300 MHz,


1H), 5.45-5.44(m, 1H), 5.38-5.29 (m, 1H), 4.66-4.64 (m, 2H), 3.90 (s, 3H), 3.80 (s, 3H); 13

C

NMR (300 MHz, CDCl3) 167.61, 150.06, 149.49, 144.72, 132.70, 127.50, 122.31, 118.34,

115.49, 112.80, 110.01, 69.68, 55.87, 51.56; ; LC MS (m/z): [M + H+ ]- 248.10

Methyl (E)-3-(4-((5-cyanopentyl) oxy)-3-methoxyphenyl) acrylate (3u)


1H NMR (300 MHz,


1H), 4.06 (t, J=5.4Hz, 6.9Hz, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 2.42-2.37 (m, 2H), 1.92-1.59 (m,

6H); 13

C NMR (300 MHz, CDCl3) 167.64, 150.48, 149.55, 144.75, 127.48, 122.48, 119.60,

115.52, 112.48, 110.16, 68.43, 55.95, 51.60, 31.79, 28.24, 25.36, 25.16, 17.07; ; LC MS (m/z):

[M + H+ ]- 303.15

Methyl (E)-3-(4-(benzyloxy)-3-methoxyphenyl) acrylate (3v)


1H NMR (300 MHz,

CDCl3): 7.61 (d, J=15.9Hz, 1H), 7.44-7.30 (m, 5H), 7.06-7.01 (m, 2H), 6.81 (d, J=8.1Hz, 1H),

6.30 (d, J=15.9Hz, 1H), 5.19 (s, 2H), 3.91 (s, 3H), 3.79 (s, 3H); 13


167.66, 150.27, 149.76, 144.76, 136.58, 128.65, 128.28, 128.03, 127.73, 127.21,122.37, 115.62,

113.43, 110.26, 70.86, 66.27, 56.00, 51.52; ; LC MS (m/z): [M + H+ ]- 298.12

Methyl (E)-3-(4-(hexadecyloxy)-3-methoxyphenyl) acrylate (3w)


1H NMR (300 MHz,


1H), 4.04 (t,J= 6.9, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 1.90-1.80 (m, 2H), 1.30 (d, J=13.5Hz, 26H),

0.89 (t, J=6.6Hz, 6.3Hz, 3H); ); 13

C NMR (300 MHz, CDCl3) 167.69, 150.81, 149.49, 144.86,

127.11, 122.55, 115.24, 112.30, 110.10, 69.01, 63.06, 55.96, 51.55, 32.80, 31.90, 29.67, 29.63,

29.56, 29.51, 29.42, 29.34, 29.01, 25.89, 25.72, 22.67, 14.09; ; LC MS (m/z): [M + H+ ]- 432.32

(E)-3-(3-methoxy-4-(((4-nitrophenyl) carbamoyl) oxy) phenyl) acrylic acid (3x)

Yield: 40%; Mp: 172-174oC; MF: C17H14N2O7; Appearance: pale yellow solid;

1H NMR (300

MHz, d6-DMSO): 12.4 (s, 1H), 11.03 (s, 1H), 8.25 (d, J=9.3Hz, 2H), 7.73 (d, J=9Hz, 2H), 7.63-

7.53 (m, 3H), 7.29 (s, 2H), 6.62 (d, J=16.2Hz, 1H), 3.85 (s, 3H); 13

C NMR (300 MHz, d6-

DMSO) 167.98, 155.65, 149.01, 147.86, 144.46, 135.59, 126.36, 125.72, 125.10, 122.76, 118,

117.97, 115.58, 115.47, 112.35, 111.08, 55.63, ; LC MS (m/z): [M + H+ ]- 358.08

1-(4-hydroxy-3-methoxyphenyl)-1,2-dihydro-3H-benzo[f]chromen-3-one (4a)

Yield : 35%; Mp: 160-162; MF: C20H16O4; Appearance: White solid; 1H NMR (300 MHz,

CDCl3): 7.83 (q, J=8.7Hz,7.2Hz, 3H), 7.46 (pd, J=1.5Hz, 2H), 7.34 (d, J=8.7Hz, 1H), 6.78 (d,

J=8.4Hz, 1H), 6.61 (q, J=1.8Hz, 3.9Hz, 2H), 5.53(s, 1H), 4.88(q, J=2.4Hz, 3.6Hz, 1H) 3.75 (s,

3H), 3.15 (t, J=6.6Hz, 3.3Hz, 2H); C=74.11%, H=4.99%, O=19.79%; ; LC MS (m/z): [M + H+ ]-

320.10

8-bromo-1-(4-hydroxy-3-methoxyphenyl)-1,2-dihydro-3H-benzo[f]chromen-3-one (4b)

Yield : 61%; Mp: 174-176; MF: C20H15BrO4; Appearance: White solid; 1H NMR (300 MHz,

CDCl3): 7.94 (d, J=1.8Hz, 1H), 7.70 (d, J=8.7Hz, 1H), 7.60 (d, J=9Hz, 1H), 7.47 (dd, J=2.1Hz,

1H), 7.29 (d, J=9Hz, 1H), 6.718 (t, J=4.2Hz,4.5Hz,1H), 6.51-6.47(m, 2H), 5.47(s, 1H), 4.71(q,

J=2.7Hz,3.6Hz, 1H), 3.69 (s, 3H), 3.16-3.02 (m, 2H); 13

C NMR (300 MHz, CDCl3):166.89,

149.87, 147.08, 145.17, 132.23, 132.11, 130.78, 130.71, 129.59, 129.0, 124.87, 119.8, 119.26,

118.73, 118.21, 114.95, 108.99, 55.86, 37.64, 37.43; C=60.1%, H=3.699%, O=15.99%; ; LC MS

(m/z): [M + H+ ]- 398.02

7-hydroxy-4-(4-hydroxy-3-methoxyphenyl) chroman-2-one (4c)

Yield : 30%; Mp: 168-170; MF: C16H14O5; Appearance: White solid; 1H NMR (300 MHz, d6-

DMSO): 9.72 (s, 1H), 8.93 (s, 1H), 6.83 (d, J=8.1Hz, 1H), 6.78(d, J=1.8Hz, 1H), 6.69 (d,

J=8.1Hz, 1H), 6.55-6.50 (m, 2H), 6.43 (dd, J=1.8Hz, 1H), 4.24 (t, J=6Hz, 6.3Hz, 1H), 3.71 (s,

3H), 3.02 (d, J=6.3Hz, 2H); 13

C NMR (300 MHz, d6-DMSO): 167.86, 157.37, 151.79, 147.63,

145.45, 132.37, 128.83, 119.27, 116.65, 115.37, 111.55, 111.48, 103.12, 55.50, 36.77, 30.59;

C=67.15%, H=5.01%, O=28.02%; ; LC MS (m/z): [M + H+ ]- 286.08

2

Table S1. In vitro anti-proliferative activity of Ferulic acid derivatives

Compound

code

IC50 value against K562 cells

( µg/mL)

IC50 value

against U937

cells

( µg/mL)

IC50 value

against HepG-

2 cells

( µg/mL)

IC50 value

against

Normal

PBMC cells

( µg/mL)

1 >100 54.68 35.07 76.54

2 >100 32.21 >100 >100

2a >100 >100 >100 >100

2b >100 >100 >100 >100

2c >100 >100 >100 >100

2d >100 >100 >100 >100

2e >100 >100 >100 >100

2f >100 16.89 >100 >100

2h 35 21.11 42.18 68.14

2i 4.02 33.14 32.94 51.32

2j 60 >100 51.01 74.18

2k >100 >100 >100 >100

3 >100 >100 >100 >100

3a >100 >100 >100 >100

3b >100 >100 >100 >100

3d >100 >100 >100 >100

3e >100 10.12 22.64 >100

3f >100 >100 >100 >100

3g >100 >100 >100 >100

3h >100 >100 >100 >100

3i >100 >100 >100 >100

3j 6.30 40.21 38.54 56.98

3k >100 >100 >100 >100 3l >100 >100 >100 >100 3m >100 >100 >100 >100 3n >100 >100 >100 >100 3o >100 >100 >100 >100 3p >100 >100 >100 >100 3q >100 >100 >100 >100 3r >100 >100 >100 >100 3s >100 >100 >100 >100 3t >100 >100 >100 >100 3u >100 19.23 34.56 79.34 3v >100 >100 >100 >100 3w >100 >100 >100 >100 3x >100 >100 >100 >100 4a >100 >100 >100 >100 4b >100 >100 >100 >100 4c >100 >100 >100 >100

Imatinib 0.1 n.d -

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